Project Summary/Abstract The two-signal model in which an antigen-presenting cell-derived costimulatory signal, epitomized by CD28, is needed for T cells to respond maximally to T cell receptor (TCR) stimulation is now part of the immunology canon and fueled exciting immunotherapies for transplant rejection, autoimmunity, and cancer. Most of the advances in the costimulation field, however, applied to conventional Foxp3neg CD4+ T cells. Although CD28 enhances the development of Foxp3+ regulatory (Treg) cells as they are selected by high affinity TCR recognition of self peptides in the thymus, and is required for their autoimmunity-suppressing functions, much less is known about the costimulatory requirements of Treg cells as they participate in immune responses to foreign peptides. During the last funding period, we found that the pre-immune repertoires of CD4+ T cells specific for foreign peptides from microbes contain both conventional AND Treg cells. Both populations underwent clonal expansion in the secondary lymphoid organs after infection with a microbe expressing the relevant peptide but the two populations formed different types of effector cells and generated memory cells with different efficiencies. Our results lead to the remarkable conclusion that two parallel CD4+ T cell repertoires exist for each foreign peptide ? one composed of conventional nave cells and a smaller one composed of Treg cells. Almost nothing is known about the signals that govern the activation of foreign peptide-specific Treg cells during immune responses to infection or their interactions with conventional T cells responding to the same peptide. The goal of this project is to determine whether foreign peptide-specific Treg cells require CD28 costimulation during immune responses to infection and if so understand the signal transduction process. We will also test the intriguing possibility that foreign peptide-specific Treg cells play special roles in regulating the expansion and differentiation of the conventional T cells specific for the same peptide as both populations respond during infection. This research could shed light on the understudied role that Treg cells play during infection and provide new uses for modulation of T cell costimulation.